ER Ca 2+ depletion activates the calcium-release activated calcium (CRAC) channel through a mechanism in which the ER Ca 2+ sensor STIM1 binds and opens the pore-forming Orai1 subunits in the plasma membrane. While STIM1 is required for opening, extracellular Ca 2+ further increases channel activity up to ∼5-fold over several seconds in a process called calcium-dependent potentiation (CDP). Conversely, removing Ca 2+ unmasks a large monovalent current that depotentiates to ∼20% of its peak over tens of seconds. CDP is a potent example of permeant ion effects on gating, responsible for up 80% of maximal channel activity, yet the mechanism is unknown. The degree of potentiation correlates with the divalent occupancy of the selectivity filter (SF), identifying the SF as the CDP binding site. The SF comprises a ring of six glutamates (E106), one from each Orai1 subunit, located directly above the hydrophobic gate (V102, F99) opened by STIM1. We hypothesize that Ca 2+ removal causes the E106 side chains to rearrange, biasing the activation gate directly below it toward closure. We monitored SF structure during depotentiation using CRAC current-voltage relations from HEK293 cells overexpressing STIM1 and Orai1. After Ca 2+ removal, the inward rectification of Ba 2+ , Na + , and Li + currents changes within the first few seconds of depotentiation, consistent with coupling between SF structure and gating. A mutation that constitutively opens the activation gate, V102A, also removes the ability of Ca 2+ to modulate channel activity, shown by elimination of CDP and depotentiation in the presence and absence of Ca 2+ , respectively. These results support our CDP mechanism: Ca 2+ constrains the SF to adopt a conformation that stabilizes the open state of the hydrophobic gate directly below it. In this case, the permeant ion serves as a structural element to optimize channel opening.
Bennett et al. (Sun,) studied this question.